Method for the microbiological production of carotenes



United States Patent P 112,642 Int. Cl. C12b 1/00; C12k 1 /10 US. Cl.195-28 9 Claims ABSTRACT OF THE DISCLOSURE 1 Carotenes aremicrobiologically produced by cultivating opposite mated strains ofBlakeslea trispora NRRL 289 and 2896 in a liquid medium in which thereis used as a lipid source, an autoclave oil having an iodine value above100. Autoclave oils are by-products which 2 are recovered from bleachingmaterials used in the refining of vegetable oils such as soybean andlinseed oil.

This invention relates to the production of carotenes by microbiologicalfermentation processes and more particularly to an improved method ofproduction of carotenes by the aerobic cultivation of sexually oppositemated strains of fungi belonging to the family Ohoanephorac/eae in aliquid aqueous medium containing normal nutrient substances such as:protein, carbohydrate and lipid sources, and also vitamins, mineralsalts and hydrocarbons, wherein a new lipid source namely oil industryby-products, which are so-called autoclave oils having an iodine valueabove 100, recovered from bleaching materials after the refining ofvegetable oils are used.

The microbiological production of carotenes by the aerobic cultivationof mated strains of fungi, such as those of the genera Choanephora andBlakeslea is well known in the art. In the known carotene production bythe cultivation of mated strains of Blakeslea trispora NRRL 2895 andNRRL 2896 lipid materials such as naturally occurring vegetable oils oranimal fats are normally added to the fermentation medium. For example,in Applied Microbiology, vol. 12, No. 2, pp. 150-154, 1964, the additionof such vegetable oils as soybean oil, cottonseed oil and corn oil isrecommended. In US. Patent No. 3,025,221, the use of choice white greaseis advised.

The addition of vegetable oils or animal fats to fermentation media inthe microbiological production of carotenes causes the stimulation ofcarotene synthesis, probably due to the fatty acid content in the lipidmaterial employed.

It has now been discovered, in accordance with the present invention,that a better effect may be attained in carotene biosynthesis by using akind of lipid material which is different from naturally occuring oilsor fats. More particularly, it has been found that specific oil industryby-products, which are obtained in oil recovering processes frombleaching material after the refining of vegetable oils, give a muchbetter and enhanced effect on carotene yield than the naturallyoccurring raw or refined vegetable oils. These by-products, which are socalled autoclave oils, contain, due to the specific adsorptionphenomenon on bleaching material, not only a very suitable fatty acidcomposition, but principally in being compared favorably with naturallyoccurring vegetable oils, they contain considerable amounts ofchlorophyll, carotenoids and related compounds, different gums or slimysubstances, andother unknown active substances which are stimulativefactors in carotene biosynthesis, which together with the containedfatty acids have an advantageous influence on the yield of carotene inthe fermentation process.

According to the invention, the addition of so-called autoclave oilswhich are oil-industry by-products obtained in the refining of vegetableoils, used at levels from about 3 percent to 6 percent of the liquidmedium, can replace the more expensive vegetable oils or animal fatswhich are several times more costly, while at the same time obtaininghigher carotene yields in fermentation processes.

The enhanced effect on the carotene yield caused by the addition ofso-called autoclave oils, which are re- 5 covered from bleachingmaterial in vegetable oil refining processes, depends on the compositionof the other nutrient substances used as adjuncts in the fermentationmedia and which comprise 540% of the media. The following examples aregiven by way of illustration and 0 are not intended as a limitation ofthis invention. Indeed,

many modifications may be made without departing from the spirit andscope thereof.

Kerosene, deodorized 4 Thiamine hydrochloride 0.0002 "Autoclave oil,recovered from bleaching material used in the refining of soybean oil orlinseed oil 4.6 Tap water The above mentioned medium is sterilized for45-60 minutes at 121 C., cooled and inoculated by adding 5-10 volumepercent of a liquid inoculum obtained by 2 days cultivation of materstrains of Blakeslea trispora NRRL 2895 and NRRL 2896 The inoculatedmedium is then incubated under aerobic conditions at 26-28 C. for 4-6days. After 48 hours incubation, there is added aseptically ozorB-ionone in an amount of 0.0935 part by weight to the fermentationmedium. After achieving the maximal carotene concentration, whichusually occurs after 5 days of fermentation, the mycelium is recoveredby filtrationfor example through a rotary filter--and dried in vacuum oron an atmospheric double drum dryer.

' The carotene preparation, obtained in the form of dried mycelium, maybe used directly for feed vitaminization or may be extracted in a knownway to obtain a carotene preparation in oil for the coloring orvitaminization of food products. The carotene yields obtained in thisfermentation process are about 95 mg./ ml. of medium.

EXAMPLE 2 The following medium is prepared:

Parts by weight 3 Yeast, dried Corn, ground whole 2.5 Soybean, groundwhole 3 Citrus molasses 5 Kerosene, deodorized 4 Thiamine hydrochloride0.0002

0 Autoclave oil, recovered from bleaching material used in the refiningof soybean oil 4.6

Tap water The sterilization and the whole production process of caroteneby fermentation is conducted in the same manner as described in Example1 through the addition of ozor fl-ionone (after 48 hours incubation).

4 EXAMPLE 5 The following medium is prepared:

Parts by weight 4 The carotene yield obtained in this fermentation proc-5 y ground Whole ess is about 100 mg./100 ml. of medium. (39m, groundWhole 2 Citrus molasses 5 EXAMPLE 3 Kerosene, deodorized 3.2

Thiamine hydrochloride 0.0002

The following medium 18 prepared: Autoclave oil, recovered frombleaching material used in the refining of soybean oil 3.7

Parts y welght Tap water 95 Cottonseed embryo meal 5 Corn, ground 'whole2- Th sterilization and the whole process of producing Citrus molasses 5carotene by fermentation is conducted in the same man- Keroscne,deodorized 4 ner as described in Exam le 1, through the addition ofThiamine hydrochloride 0.0002 1- r fl-ionon Autoclave recovered frombleachlhg The carotene yields obtained in this fermentation procterialused in the refining of soybean oil 4.6 655 i about 95 mg /10() m1, ofmediurn Tap water 95 EXAMPLE 6 The sterilization and the whole processfor producing carotene by fermentation is conducted in the same man- Thefollowing medium 15 prepared ner as described in Example 1, through theaddition of Parts by weight aor fl-ionone. Yeast, dried 2 The caroteneyield obtained in this fermentation proc- Soybean, ground whole 2 ess isabout 100 mg./100 ml. of medium. Corn, ground whole 2 Citrus molasses 5EXAMPLE 4 Kerosene, deodorized 3.2

Thiamine hydrochloride 0.0002

Parts by Welght Autoclave oil, recovered from bleaching macottonseedembryo meal 5 terial used in the refining of soybean oil 3.7

Corn, ground whole 2.5 Tap water 95 Kerosene, deodorized 4 Thiaminehydrochloride 0.0002 The sterilization and the whole process ofproducing Autoclave L recovered from bleachlhg carotene are conducted inthe same manner as described terial used in the refining of soybean oil4.6 in Example 1, through the addition of aor B-ionone. Tap water 95 Thecarotene yield obtained in this fermentation process is about 100 mg./100 ml. of medium.

The Sterlhlatlofi f the Whole p f pl'oduclhg 40 Comparative fermentationexperiments were made on Cawtehe y fermehtatloh 1S conducted in the Samea laboratory scale in shaken-flask cultures, using various ner asdescribed in Example 1. medium components including different autoclaveoils The carotene yield obtained in this fermentation procand refinedsoybean oil. The obtained carotene yields are ess are about 95 mg./100ml. of medium. summarized in Table 1.

TABLE 1 Carotene Dried conceu- Carotene The basic mycelium tration yieldin medium The kind of in g. per in dried mg. per Yield formula as instimulating 100ml. mycclium 100ml. coefficient example agent The kind oflipid source medium in mg./g. medium in percent fl-Iouonc.Boybeanoil,refined 7.0 10.4 72.8 100 1 do... "Apfoclavg oil, recoveredfrom bleaching material used in refining 7. 0 11. 95 83. 114. 9

O msee O1 Autoclave oil, recovered from bleaching material used inrefining 7. 05 13. 7 96. 6 132. 7

of soybean oil.

Soybean oil, refined 7. 7 10. 25 91. 6 100 Autoclave oil, recovered frombleaching material used in refining 7. 13. 1 102. 8 112. 1

of soybean oil.

Soybean oil, refined 7. 5 13. 75 103. 1 100 "Autoclave oil, recoveredfrom bleaching material used in refining 6. 25 17. 25 107. 8 104. 6

of soybean oil.

Soybean oil, refined 6. 2 11. 45 71 100 Autoclave oil, recovered frombleaching material used in refining 6. 1 14. 5 88.45 124. 6

of soybean oil.

Soybean oil, refined. 6. 1 6. 0 36. 6 100 "Autoclave oil recovered frombleaching material used in refining 6. 15 9. 9 60. 9 166. 4

of soybean oil.

5 Citrus molasses- Soybean oil, refined 6.6 13.4 88.4 100 5 do"Autoclave oil,"recoveredirom bleachingrnaterialusedinrefining 6.4 14.7594.4 106.8

of soybean oil.

6 Citrus molasses- Soybean oil,reflned 6.9 14.5 100.05 1 6 do 6. 6 15.75 103. 103. 9

of soybean o "Autoclave 011,1 recovered from bleaching material used inrefining We claim:

1. A process for the production of carotene comprising cultivating fungiof the family Choanephoraceae in a liquid medium containing protein andcarbohydrate sources and about 3-6% by weight of the liquid medium of anautoclave oil, having an iodine value above 100, said autoclave oilbeing a by-product recovered from bleaching materials which are used inthe refining of vegetable oils, and recovering the carotene.

2. A process as claimed in claim 1, wherein the fungi include oppositemated strains.

3. A process as claimed in claim 2, wherein the fungi are Blwkesleatrisopora NRRL 2895 and NRRL 2896 4. A process as claimed in claim 3,wherein the medium further contains a hydrocarbon and a vitamin.

5. A process as claimed in claim 4, wherein the fungi are introducedinto the medium in the form of a liquid inoculum which is added at about5-10 percent by volume to the medium whereafter the medium is incubatedunder aerobic conditions at about 26-28 C. for about 4-6 days.

6. A process as claimed in claim 5, wherein the hydrocarbon is keroseneand the vitamin is thiamine hydrochloride.

7. A method for the production of carotene comprising cultivatingoppositely mated strains of the fungi, Blakeslea trispora NRRL 2895 andNRRL 2896 in a liquid medium containing protein and carbohydrate sourcesand further containing a lipid source which is an References CitedUNITED STATES PATENTS 3/1962 Ciegler et a1. -28 3,291,701 12/1966 Fulde19528 ALVIN E. TANENHOLTZ, Primary Examiner US. Cl. X.R.

